These projects are designed to further our knowledge of chromatin replication. Histone synthesis and deposition are major aspects of this replication, as these proteins are required to package DNA such that it is accessible to effector molecules and polymerases. Studies on these processes have been initiated by synchronizing HTC cells and density labeling the DNA with iododeoxyuridine. These studies indicate that the synthesis and deposition of histones H3 and H4 is rigorously controlled, such that deposition occurs only on newly replicated DNA. When the limited synthesis of these histones occurs in G[unreadable]1[unreadable], these histones are prevented from associating with DNA until the onset of DNA synthesis (S phase) and then specifically on the newly replicated DNA. Histones H2A and H2B deposit on either G[unreadable]1[unreadable] DNA or S phase DNA at a rate suggestive of constant exchange. When deposition is occurring in S phase, H2A and H2B deposit on both newly replicated and nonreplicated DNA. Preliminary evidence suggests that the site for this nonreplicated deposition is the DNA actively involved in RNA transcription. The histone variants X and Z appear to deposit primarily on nonreplicated DNA, as we cannot detect selective deposition of these histones on newly replicated DNA. In addition, by using an alternative procedure of density labeling histones with dense amino acids ([unreadable]15[unreadable]N, [unreadable]13[unreadable]C, [unreadable]2[unreadable]H) and subsequently crosslinking the histones to each other within the nucleosome to form an octameric complex, we have established that during the deposition of newly synthesized histones tetrameric forms of new H3-H4 and dimeric forms of new H2A-H2B remain a stable part of the nucleosome throughout the cell cycle and into the next replication events. Thus the movement of histone at the replication fork necessitates that these structures be maintained even though when these histones redeposit on the daughter strands during the second replication event they do so in a random way. There is, therefore, both a dynamic and static nature to histone mobility at the replication fork and at active or inactive genes. (K)